Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers

ABSTRACT

Hydrogels of polymerized and crosslinked macromers comprising hydrophilic oligomers having biodegradable monomeric or oligomeric extensions, which biodegradable extensions are terminated on free ends with end cap monomers or oligomers capable of polymerization and cross linking are described. The hydrophilic core itself may be degradable, thus combining the core and extension functions. Macromers are polymerized using free radical initiators under the influence of long wavelength ultraviolet light, visible light excitation or thermal energy. Biodegradation occurs at the linkages within the extension oligomers and results in fragments which are non-toxic and easily removed from the body. Preferred applications for the hydrogels include prevention of adhesion formation after surgical procedures, controlled release of drugs and other bioactive species, temporary protection or separation of tissue surfaces, adhering of sealing tissues together, and preventing the attachment of cells to tissue surfaces.

This application is a continuation of U.S. application Ser. No.09/492,011 filed Jan. 26, 2000, entitled “PhotopolymerizableBiodegradable Hydrogels as Tissue Contacting Materials andControlled-Release Carriers”, by Jeffrey A. Hubbell, Chandrashekhar P.Pathak, Amarpreet S. Sawhney, Neil P. Desai, and Jennifer L. Hill, nowU.S. Pat. No. 6,306,922, which is a continuation of U.S. Ser. No.09/128,917 filed Aug. 4, 1998, now U.S. Pat. No. 6,060,582, which is acontinuation of U.S. Ser. No. 08/700,237 filed Aug. 20, 1996, now U.S.Pat No. 5,986,043, which is a divisional of U.S. Ser. No. 08/468,364filed Jun. 6, 1995, now U.S. Pat. No. 5,567,435, which is a divisionalof U.S. Ser. No. 08/379,848 filed Jan. 27, 1995, now U.S. Pat. No.5,626,863, which is a divisional of U.S. Ser. No. 08/022,687 filed Mar.1, 1993, now U.S. Pat. No. 5,410,016, which is a continuation-in-part ofU.S. Ser. No. 07/843,485 filed Feb. 28, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates to photopolymerizable biodegradablehydrogels for use as tissue adhesives and in controlled drug delivery.

BACKGROUND OF THE INVENTION

This is a continuation-in-part of U.S. patent application Ser. No.07/843,485, filed Feb. 28, 1992, entitled “PhotopolymerizableBiodegradable Hydrogels as Tissue Contacting Materials and ControlledRelease Carriers” by Jeffrey A. Hubbell, Chandrashekhar P. Pathak, andAmarpreet S. Sawhney.

Hydrogels as Controlled-release Carriers

Biodegradable hydrogels can be carriers for biologically activematerials such as hormones, enzymes, antibiotics, antineoplastic agents,and cell suspensions. Temporary preservation of functional properties ofa carried species, as well as controlled release of the species intolocal tissues or systemic circulation, are possible. Proper choice ofhydrogel macromers can produce membranes with a range of permeability,pore sizes and degradation rates suitable for a variety of applicationsin surgery, medical diagnosis and treatment.

Adhesives and Sealers

Fibrin gels have been used extensively in Europe as sealants andadhesives in surgery (Thompson et al., 1988, “Fibrin Glue: A review ofits preparation, efficacy, and adverse effects as a topical hemostat,”Drug Intell. and Clin. Pharm., 22:946, Gibble et al., 1990, (1990),“Fibrin glue: the perfect operative sealant?” Transfusion, 30(8) :741).However, they have not been used extensively in the United States due toconcerns relating to disease transmission from blood products. Syntheticpolymers have been explored as adhesives (Lipatova, 1986, “Medicalpolymer adhesives,” Advances in Polymer Science 79:65-93), but thesematerials have been associated with local inflammation, cytotoxicity,and poor biocompatibility.

Prevention of Postoperative Adhesions

Formation of post-surgical adhesions involving organs of the peritonealcavity and the peritoneal wall is a frequent and undesirable result ofabdominal surgery. Surgical trauma to the tissue caused by handling anddrying results in release of a serosanguinous (proteinaceous) exudatewhich tends to collect in the pelvic cavity (Holtz, G., 1984). If theexudate is not absorbed or lysed within this period it becomes ingrownwith fibroblasts, and subsequent collagen deposition leads to adhesionformation.

Numerous approaches to elimination of adhesion formation have beenattempted, with limited success in most cases. Approaches have includedlavage of the peritoneal cavity, administration of pharmacologicalagents, and the application of barriers to mechanically separatetissues. For example, Boyers et al., (1988) “Reduction of postoperativepelvic adhesions in the rabbit with Gore-Tex surgical membrane,” Fertil.Steril., 49:1066, examined Gore-Tex surgical membranes in the preventionof adhesions. For a review of adhesion prevention, see Holtz (1984)“Prevention and management of peritoneal adhesions,” Fertil. Steril.,41:497-507. However, none of these approaches has been cost effectiveand effective in in vivo studies.

Solutions of Poloxamer 407 have been used for the treatment ofadhesions, with some success. Poloxamer is a copolymer of ethylene oxideand propylene oxide and is soluble in water; the solutions are liquidsat room temperature. Steinleitner et al. (1991) “Poloxamer 407 as anIntraperitoneal Barrier Material for the Prevention of PostsurgicalAdhesion Formation and Reformation in Rodent Models for ReproductiveSurgery,” Obstetrics and Gynecology, 77 (1):48 and Leach et al. (1990)“Reduction of postoperative adhesions in the rat uterine horn model withpoloxamer 407, Am. J. Obstet. Gynecol., 162(5):1317, examined Poloxamersolutions in peritoneal adhesion models and observed statisticallysignificant reductions in adhesions; however, they were unable toeliminate adhesions, perhaps because of limited adhesion and retentionon the injury site.

Oxidized regenerated cellulose has been used extensively to preventadhesions and is an approved clinical product, trade-named InterceedTC7. This barrier material has been shown to be somewhat effective inrabbits (Linsky et al., 1987 “Adhesion reduction in a rabbit uterinehorn model using TC-7, ” J. Reprod. Med., 32:17; Diamond et al., 1987“Pathogenesis of adhesions formation/reformation: applications toreproductive surgery,” Microsurgery, 8:103) and in humans (Interceed(TC7) Adhesion Barrier Study Group, 1989). It was shown to be moreeffective if pretreated with heparin, but was still unable to completelyeliminate adhesions (Diamond et al., 1991 “Synergistic effects ofINTERCEED (TC7) and heparin in reducing adhesion formation in the rabbituterine horn model,” Fertility and Sterility, 55(2):389).

In summary, several lavage/drug/material approaches have been explored,but none of these approaches has been able to eliminate adhesions. Anideal material barrier would not evoke an adhesion response itself, stayin place without suturing (Holtz et al., 1982 “Adhesion induction bysuture of varying tissue reactivity and caliber,” Int. J. Fert.,27:134), degrade over a few weeks', time, effectively reduce adhesionsto very low extent, and be capable of delivering a drug to the localsite of application for several days' time. None of the approachesdeveloped and described to date meet these requirements.

Synthetic Biodegradable Polymers

The field of biodegradable polymers has developed rapidly since thesynthesis and biodegradability of polylactic acid was first reported byKulkarni et al., 1966 “Polylactic acid for surgical implants,” Arch.Surg., 93:839. Several other polymers are known to biodegrade, includingpolyanhydrides and polyorthoesters, which take advantage of labilebackbone linkages, as reported by Domb et al., 1989 Macromolecules,22:3200; Heller et al., 1990 Biodegradable Polymers as Drug DeliverySystems, Chasin, M. and Langer, R., Eds., Dekker, New York, 121-161.Since it is desirable to have polymers that degrade into naturallyoccurring materials, polyaminoacids have been synthesized, as reportedby Miyake et al., 1974, for in vivo use. This was the basis for usingpolyesters (Holland et al., 1986 Controlled Release, 4:155-180) ofα-hydroxy acids (viz., lactic acid, glycolic acid), which remain themost widely used biodegradable materials for applications ranging fromclosure devices (sutures and staples) to drug delivery systems (U.S.Pat. No. 4,741,337 to Smith et al.; Spilizewski et al., 1985 “The effectof hydrocortisone loaded poly(dl-lactide) films on the inflammatoryresponse, ” J. Control. Rel 2:197-203).

The time required for a polymer to degrade can be tailored by selectingappropriate monomers. Differences in crystallinity also alterdegradation rates. Due to the relatively hydrophobic nature of thesepolymers, actual mass loss only begins when the oligomeric fragments aresmall enough to be water soluble. Hence, initial polymer molecularweight influences the degradation rate.

Degradable polymers containing water-soluble polymer elements have beendescribed. Sawhney et al., (1990) “Rapidly degraded terpolymers ofdl-lactide, glycolide, and ε-caprolactone with increased hydrophilicityby copolymerization with polyethers,” J. Biomed. Mater. Res.24:1397-1411, copolymerized lactide, glycolide and ε-caprolactone withPEG to increase its hydrophilicity and degradation rate. U.S. Pat. No.4,716,203 to Casey et al. (1987) synthesized a PGA-PEG-PGA blockcopolymer, with PEG content ranging from 5-25% by mass. U.S. Pat. No.4,716,203 to Casey et al. (1987) also reports synthesis of PGA-PEGdiblock copolymers, again with PEG ranging from 5-25%. U.S. Pat. No.4,526,938 to Churchill et al. (1985) described noncrosslinked materialswith MW in excess of 5,000, based on similar compositions with PEG;although these materials are not water soluble. Cohn et al. (1988) J.Biomed. Mater. Res. 22:993-1009 described PLA-PEG copolymers that swellin water up to 60%; these polymers also are not soluble in water, andare not crosslinked. The features that are common to these materials isthat they use both water-soluble polymers and degradable polymers, andthat they are insoluble in water, collectively swelling up to about 60%.

Degradable materials of biological origin are well known, for example,crosslinked gelatin. Hyaluronic acid has been crosslinked and used as adegradable swelling polymer for biomedical applications (U.S. Pat. No.4,987,744 to della Valle et al., U.S. Pat. No. 4,957,744 to Della Valleet al. (1991) “Surface modification of polymeric biomaterials forreduced thrombogenicity,” Polym. Mater. Sci. Eng., 62:731-735]).

Use of Biodegradable Materials for Controlled Drug Release

Most hydrophilic drugs are mechanically dispersed as suspensions withinsolutions of biodegradable polymers in organic solvents. Protein andenzyme molecular conformations are frequently different under thesecircumstances than they would be in aqueous media. An enzyme dispersedin such a hydrophobic matrix is usually present in an inactiveconformation until it is released into the surrounding aqueousenvironment subsequent to polymer degradation. Additionally, someproteins may be irreversibly denatured by contact with organic solventsused in dispersing the protein within the polymer.

Polymer Synthesis, Degradation and Local Synthesis

Rapidly-degrading polymers currently suggested for short-termmacromolecular drug release may raise local concentrations ofpotentially hazardous acidic degradation byproducts. Further, allbiodegradable synthetic polymers reported thus far can only be processedin organic solvents and all biodegradable polymers are synthesized underconditions which are not amenable to polymerization in vivo. Thus, ithas not been possible to make implantable materials as preciselyconformed barriers, shaped articles, or membranes capable of deliveringbioactive materials to the local tissue.

It is therefore an object of the present invention to provide hydrogelswhich are biocompatible, biodegradable, and can be rapidly formed bypolymerization in vivo.

It is a further object of the present invention to provide a macromersolution which can be administered during surgery or outpatientprocedures and polymerized as a tissue adhesive, tissue encapsulatingmedium, tissue support, or drug delivery medium.

It is a still further object of the present invention to provide amacromer solution which can be polymerized in vivo in a very short timeframe and in very thin, or ultrathin, layers.

SUMMARY OF THE INVENTION

Disclosed herein are biocompatible, biodegradable, polymerizable and atleast substantially water soluble macromers, having a variety of uses invivo. The macromers include at least one water soluble region, at leastone region which is biodegradable, usually by hydrolysis, and at leasttwo free radical-polymerizable regions. The regions can, in someembodiments, be both water soluble and biodegradable. The macromers arepolymerized by exposure of the polymerizable regions to free radicalsgenerated, for example, by photosensitive chemicals and dyes.

An important aspect of the macromers are that the polymerizable regionsare separated by at least one degradable region to facilitate uniformdegradation in vivo. There are several variations of these polymers. Forexample, the polymerizable regions can be attached directly todegradable extensions or indirectly via water soluble nondegradablesections so long as the polymerizable regions are separated by adegradable section. For example, if the macromer contains a simple watersoluble region coupled to a degradable region, one polymerizable regionmay be attached to the water soluble region and the other attached tothe degradable extension or region. In another embodiment, the watersoluble region forms the central core of the macromer and has at leasttwo degradable regions attached to the core. At least two polymerizableregions are attached to the degradable regions so that, upondegradation, the polymerizable regions, particularly in the polymerizedgel form, are separated. Conversely, if the central core of the macromeris formed by a degradable region, at least two water soluble regions canbe attached to the core and polymerizable regions attached to each watersoluble region. The net result will be the same after gel formation andexposure to in vivo degradation conditions. In still another embodiment,the macromer has a water soluble backbone region and a degradable regionaffixed to the macromer backbone. At least two polymerizable regions areattached to the degradable regions, so that they are separated upondegradation, resulting in gel product dissolution. In a furtherembodiment, the macromer backbone is formed of a nondegradable backbonehaving water soluble regions as branches or grafts attached to thedegradable backbone. Two or more polymerizable regions are attached tothe water soluble branches or grafts. In another variation, the backbonemay be star shaped, which may include a water soluble region, abiodegradable region or a water soluble region which is alsobiodegradable. In this general embodiment, the star region containseither water soluble or biodegradable branches or grafts withpolymerizable regions attached thereto. Again, the polymerizable regionsmust be separated at some point by a degradable region.

Examples of these macromers are PEG-oligoglycolyl-acrylates. The choiceof appropriate end caps permits rapid polymerization and gelation;acrylates were selected because they can be polymerized using severalinitiating systems, e.g., an eosin dye, by brief exposure to ultravioletor visible light. The poly(ethyleneglycol) or PEG central structuralunit (core) was selected on the basis of its high hydrophilicity andwater solubility, accompanied by excellent biocompatibility. A shortoligo or poly(α-hydroxy acid), such as polyglycolic acid, was selectedas a preferred chain extension because it rapidly degrades by hydrolysisof the ester linkage into glycolic acid, a harmless metabolite. Althoughhighly crystalline polyglycolic acid is insoluble in water and mostcommon organic solvents, the entire macromer is water-soluble and can berapidly gelled into a biodegradable network while in contact withaqueous tissue fluids. Such networks can be used to entrap andhomogeneously disperse water-soluble drugs and enzymes and to deliverthem at a controlled rate. Further, they may be used to entrapparticulate suspensions of water-insoluble drugs. Other preferred chainextensions are polylactic acid, polycaprolactone, polyorthoesters, andpolyanhydrides. Polypeptides may also be used. Such “polymeric” blocksshould be understood to include timeric, trimeric, and oligomericblocks.

These materials are particularly useful for controlled drug delivery,especially of hydrophilic materials, since the water soluble regions ofthe polymer enable access of water to the materials entrapped within thepolymer. Moreover, it is possible to polymerize the macromer containingthe material to be entrapped without exposing the material to organicsolvents. Release may occur by diffusion of the material from thepolymer prior to degradation and/or by diffusion of the material fromthe polymer as it degrades, depending upon the characteristic pore sizeswithin the polymer, which is controlled by the molecular weight betweencrosslinks and the crosslink-density. Deactivation of the entrappedmaterial is reduced due to the immobilizing and protective effect of thegel and catastrophic burst effects associated with othercontrolled-release systems are avoided. When the entrapped material isan enzyme, the enzyme can be exposed to substrate while the enzyme isentrapped, provided the gel proportions are chosen to allow thesubstrate to permeate the gel. Degradation of the polymer facilitateseventual controlled release of free macromolecules in vivo by gradualhydrolysis of the terminal ester linkages.

An advantage of these macromers are that they can be polymerized rapidlyin an aqueous surrounding. Precisely conforming, semi-permeable,biodegradable films or membranes can thus be formed on tissue in situ toserve as biodegradable barriers, as carriers for living cells or otherbiologically active materials, and as surgical adhesives. In aparticularly preferred embodiment, the macromers are applied to tissuehaving bound thereto an initiator, and polymerized to form ultrathincoatings. This is especially useful in forming coatings on the inside oftissue lumens such as blood vessels where there is a concern regardingrestenosis, and in forming tissue barriers during surgery which therebyprevent adhesions from forming.

Examples demonstrate the use of these macromers and polymers for theprevention of postoperative surgical adhesions in rat cecum and rabbituterine horn models. The polymer shows excellent biocompatibility, asseen by a minimal fibrous overgrowth on implanted samples. Hydrogels forthe models were gelled in situ from water-soluble precursors by briefexposure to long wavelength ultraviolet (LWUV) light, resulting information of an interpenetrating network of the hydrogel with theprotein and glycosaminoglycan components of the tissue. The degradablehydrogel was very effective, both by itself and in combination with tPA,in preventing adhesions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically illustrated macromers of the presentinvention where ______ is a water soluble core such as PEG; ˜˜˜˜˜ is ahydrolyzably degradable extension such as a polyglycolide; ====== is apolymerizable end cap or side chain such as an acrylate; and ------ is awater-soluble and hydrolyzable portion such as a hyaluronate.

FIG. 2 shows the degree of photopolymerization (dp) calculated and foundby NMR.

FIG. 3A shows the release of BSA from a PEG 1 K (1000 molecular weightPEG) glycolide diacrylate with glycolide extensions (1 KG) hydrogel intoPBS.

FIG. 3B shows release of lysozyme from PEG 18.5 K-DL-lactidetretraacrylate (18.5 KL) into PBS.

FIG. 4A shows release of active recombinant tPA from a PEG 1 K lactidediacrylate (1 KL) hydrogel.

FIG. 4B shows release of active recombinant t-PA from PEG 4 K glycolidediacrylate (4 KG) hydrogel.

FIG. 4C shows release of active recombinant tPA from a PEG 18.5K-glycolide diacrylate (18.5 KG) hydrogel into PBS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Disclosed herein are water soluble, biodegradable polymers formed frommacromers containing both water soluble regions as well as biodegradableregions and at least two regions which are polymerizable by free radicalinitiation, preferably by photopolymerization using visible or longwavelength ultraviolet radiation.

The macromers.

In general terms, the macromers are polymers that are soluble in aqueoussolutions, or nearly aqueous solutions, such as water with addeddimethylsulfoxide. They have three components including a biodegradableregion, preferably hydrolyzable under in vivo conditions, a watersoluble region, and at least two polymerizable regions. Examples ofthese structures are shown in FIG. 1.

Structure A in FIG. 1 shows a macromer having a water soluble region(______), a water soluble and degradable component (------) appended toone another. Each has a polymerizable end cap (======). Structure Bshows a major water soluble component or core region (______) extendedat either end by a degradable or hydrolyzable component (˜˜˜˜˜˜) andterminated by, at either end, a polymerizable component (======)Structure C shows a central degradable or hydrolyzable component(˜˜˜˜˜˜) bound to a water soluble component (______) capped at eitherend by a polymerizable component (======). Structure D shows a centralwater soluble component (______) with numerous branches of hydrolyzablecomponents (˜˜˜˜˜˜), each hydrolyzable component being capped with apolymerizable component (======). Structure E shows a centralbiodegradable, hydrolyzable component (˜˜˜˜˜˜) with three water solublebranches (______), each water soluble branch being capped by apolymerizable component (======) Structure F shows a long central watersoluble and hydrolyzable component (------), each end being capped by apolymerizable component (======) Structure G shows a central watersoluble and hydrolyzable component (------) capped at both ends by ahydrolyzable component (˜˜˜˜˜˜), each hydrolyzable component beingcapped by a polymerizable component (======). Structure H shows acentral water soluble and degradable or hydrolyzable component (------)with end caps or branches of a polymerizable component (======).Structure I shows a central water soluble component (______) in circularform with water soluble branches extended by a hydrolyzable component(˜˜˜˜˜˜) capped by a polymerizable component (======) Lastly, StructureJ in FIG. 1 shows a circular water soluble core component (______) withdegradable branches (˜˜˜˜˜˜), each being capped by a polymerizablecomponent (˜˜˜˜˜˜).

The various structures shown in FIG. 1 are exemplary only. Those skilledin the art will understand many other possible combinations which couldbe utilized for the purposes of the present invention.

Used herein is the term “at least substantially water soluble.” This isindicative that the solubility should be at least about 1 g/100 ml ofaqueous solution or in aqueous solution containing small amounts oforganic solvent, such as dimethylsulfoxide. By the term “polymerizable”is meant that the regions have the capacity to form additional covalentbonds resulting in macromer interlinking, for example, carbon-carbondouble bonds of acrylate-type molecules. Such polymerization ischaracteristically initiated by free-radical formation, for example,resulting from photon absorption of certain dyes and chemical compoundsto ultimately produce free-radicals.

In a preferred embodiment, a hydrogel begins with a biodegradable,polymerizable, macromer including a core, an extension on each end ofthe core, and an end cap on each extension. The core is a hydrophilicpolymer or oligomer; each extension is a biodegradable polymer oroligomer; and each end cap is an oligomer, dimer or monomer capable ofcross-linking the macromers. In a particularly preferred embodiment, thecore includes hydrophilic poly(ethylene glycol) oligomers of molecularweight between about 400 and 30,000 Da; each extension includesbiodegradable poly (α-hydroxy acid) oligomers of molecular weightbetween about 200 and 1200 Da; and each end cap includes anacrylate-type monomer or oligomer (i.e., containing carbon-carbon doublebonds) of molecular weight between about 50 and 200 Da which are capableof cross-linking and polymerization between copolymers. Morespecifically, a preferred embodiment incorporates a core consisting ofpoly(ethylene glycol) oligomers of molecular weight between about 8,000and 10,000 Da; extensions consisting of poly(lactic acid) oligomers ofmolecular weight about 250 Da; and end caps consisting acrylate moietiesof about 100 Da molecular weight.

Those skilled in the art will recognize that oligomers of the core,extensions and end caps may have uniform compositions or may becombinations of relatively short chains or individual species whichconfer specifically desired properties on the final hydrogel whileretaining the specified overall characteristics of each section of themacromer. The lengths of oligomers referred to herein may vary from twomers to many, the term being used to distinguish subsections orcomponents of the macromer from the complete entity.

Water soluble regions.

In preferred embodiments, the core water soluble region can consist ofpoly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propyleneoxide) block copolymers, polysaccharides orcarbohydrates such as hyaluronic acid, dextran, heparan sulfate,chondroitin sulfate, heparin, or alginate, proteins such as gelatin,collagen, albumin, ovalbumin, or polyamino acids.

Biodegradable regions.

The biodegradable region is preferably hydrolyzable under in vivoconditions. For example, hydrolyzable group may be polymers andoligomers of glycolide, lactide, ε-caprolactone, other hydroxy acids,and other biologically degradable polymers that yield materials that arenon-toxic or present as normal metabolites in the body. Preferredpoly(α-hydroxy acid)s are poly(glycolic acid), poly(DL-lactic acid) andpoly(L-lactic acid). Other useful materials include poly(amino acids),poly(anhydrides), poly(orthoesters), poly(phosphazines) andpoly(phosphoesters). Polylactones such as poly(ε-caprolactone),poly(ε-caprolactone), poly(δ-valerolactone) andpoly(gamma-butyrolactone), for example, are also useful. Thebiodegradable regions may have a degree of polymerization ranging fromone up to values that would yield a product that was not substantiallywater soluble. Thus, monomeric, dimeric, trimeric, oligomeric, andpolymeric regions may be used.

Biodegradable regions can be constructed from polymers or monomers usinglinkages susceptible to biodegradation, such as ester, peptide,anhydride, orthoester, phosphazine and phosphoester bonds.

Polymerizable regions.

The polymerizable regions are preferably polymerizable byphotoinitiation by free radical generation, most preferably in thevisible or long wavelength ultraviolet radiation. The preferredpolymerizable regions are acrylates, diacrylates, oligoacrylates,methacrylates, dimethacrylates, oligomethoacrylates, or otherbiologically acceptable photopolymerizable groups.

Other initiation chemistries may be used besides photoinitiation. Theseinclude, for example, water and amine initiation schemes with isocyanateor isothiocyanate containing macromers used as the polymerizableregions.

Photoinitiators and/or Catalysts.

Useful photoinitiators are those which can be used to initiate by freeradical generation polymerization of the macromers without cytotoxicityand within a short time frame, minutes at most and most preferablyseconds. Preferred dyes as initiators of choice for LWUV or visiblelight initiation are ethyl eosin 2,2-dimethoxy-2-phenyl acetophenone,other acetophenone derivatives, and camphorquinone. In all cases,crosslinking and polymerization are initiated among macromers by alight-activated free-radical polymerization initiator such as2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl eosin (10⁻⁴to 10⁻² M) and triethanol amine (0.001 to 0.1 M), for example.

The choice of the photoinitiator is largely dependent on thephotopolymerizable regions. For example, when the macromer includes atleast one carbon-carbon double bond, light absorption by the dye causesthe dye to assume a triplet state, the triplet state subsequentlyreacting with the amine to form a free radical which initiatespolymerization. Preferred dyes for use with these materials includeeosin dye and initiators such as 2,2-dimethyl-2-phenylacetophenone,2-methoxy-2-phenylacetophenone, and camphorquinone. Using suchinitiators, copolymers may be polymerized in situ by long wavelengthultraviolet light or by laser light of about 514 nm, for example.

Initiation of polymerization is accomplished by irradiation with lightat a wavelength of between about 200-700 nm, most preferably in the longwavelength ultraviolet range or visible range, 320 nm or higher, mostpreferably about 514 nm or 365 nm

There are several photooxidizable and photoreducible dyes that may beused to initiate polymerization. These include acridine dyes, forexample, acriblarine; thiazine dyes, for example, thionine; xanthinedyes, for example, rose bengal; and phenazine dyes, for example,methylene blue. These are used with cocatalysts such as amines, forexample, triethanolamine; sulphur compounds, for example, RSO₂R¹;heterocycles, for example, imidazole; enolates; organometallics; andother compounds, such as N-phenyl glycine. Other initiators includecamphorquinones and acetophenone derivatives.

Thermal polymerization initiator systems may also be used. Such systemsthat are unstable at 37° C. and would initiate free radicalpolymerization at physiological temperatures include, for example,potassium persulfate, with or without tetraamethyl ethylenediamine;benzoylperoxide, with or without triethanolamine; and ammoniumpersulfate with sodium bisulfite.

Applications for the Macromers.

Prevention of Surgical Adhesions.

A preferred application is a method of reducing formation of adhesionsafter a surgical procedure in a patient. The method includes coatingdamaged tissue surfaces in a patient with an aqueous solution of alight-sensitive free-radical polymerization initiator and a macromersolution as described above. The coated tissue surfaces are exposed tolight sufficient to polymerize the macromer. The light-sensitivefree-radical polymerization initiator may be a single compound (e.g.,2,2-dimethoxy-2-phenyl acetophenone) or a combination of a dye and acocatalyst (e.g., ethyl eosin and triethanol amine).

Controlled drug delivery.

A second preferred application concerns a method of locally applying abiologically active substance to tissue surfaces of a patient. Themethod includes the steps of mixing a biologically active substance withan aqueous solution including a light-sensitive free-radicalpolymerization initiator and a macromer as described above to form acoating mixture. Tissue surfaces are coated with the coating mixture andexposed to light sufficient to polymerize the macromer. The biologicallyactive substance can be any of a variety of materials, includingproteins, carbohydrates, nucleic acids, and inorganic and organicbiologically active molecules. Specific examples include enzymes,antibiotics, antineoplastic agents, local anesthetics, hormones,antiangiogenic agents, antibodies, neurotransmitters, psychoactivedrugs, drugs affecting reproductive organs, and oligonucleotides such asantisense oligonucleotides.

In a variation of the method for controlled drug delivery, the macromersare polymerized with the biologically active materials to formmicrospheres or nanoparticles containing the biologically activematerial. The macromer, photoinitiator, and agent to be encapsulated aremixed in an aqueous mixture Particles of the mixture are formed usingstandard techniques, for example, by mixing in oil to form an emulsion,forming droplets in oil using a nozzle, or forming droplets in air usinga nozzle. The suspension or droplets are irradiated with a lightsuitable for photopolymerization of the macromer.

Tissue Adhesives.

Another use of the polymers is in a method for adhering tissue surfacesin a patient. The macromer is mixed with a photoinitiator orphotoinitiator/cocatalyst mixture to form an aqueous mixture and themixture is applied to a tissue surface to which tissue adhesion isdesired. The tissue surface is contacted with the tissue with whichadhesion is desired, forming a tissue junction. The tissue junction isthen irradiated until the macromers are polymerized.

Tissue Coatings.

In a particularly preferred application of these macromers, an ultrathincoating is applied to the surface of a tissue, most preferably the lumenof a tissue such as a blood vessel. One use of such a coating is in thetreatment or prevention of restenosis, abrupt reclosure, or vasospasmafter vascular intervention. The photoinitiator is applied to thesurface of the tissue, allowed to react, adsorb or bond to tissue, theunbound photoinitiator is removed by dilution or rinsing, and themacromer solution is applied and polymerized. As demonstrated below,this method is capable of creating uniform polymeric coating of betweenone and 500 microns in thickness, most preferably about twenty microns,which does not evoke thrombosis or localized inflammation.

Tissue Supports.

The macromers can also be used to create tissue supports by formingshaped articles within the body to serve a mechanical function. Suchsupports include, for example, sealants for bleeding organs, sealantsfor bone defects and space-fillers for vascular aneurisms. Further, suchsupports include strictures to hold organs, vessels or tubes in aparticular position for a controlled period of time.

The following examples are presented to describe preferred embodimentsand utilities of the present invention and are not meant to limit theinvention unless otherwise stated in the claims appended hereto. Takentogether, the examples illustrate representative demonstrations of thebest mode of implementing the invention as currently understood.

Table 1 shows the code names of the various macromers synthesized in orfor use in the examples, along with their composition in terms of themolecular weight of the central PEG segment and the degree ofpolymerization of the degradable comonomer.

TABLE 1 Macromer Molecular Weight and Composition. PEG D.P. of molecularconomoner Polymer weight Comonomer per OH group Code 20,000 glycolide15    20 KG 18,500 glycolide   2.5 18.5 K 10,000 glycolide 7   10 KG 6,000 glycolide 5   6 KG  4,000 glycolide 5   4 KG  1,000 glycolide 2  1 KG 20,000 DL-lactide 10    20 KL 18,500 DL-lactide 10  18.5 KL10,000 DL-lactide 5   10 KL  6,000 DL-lactide 5   6 KL  1,000 DL-lactide2    1 KL   600 DL-lactide 2  0.6 KL   600 DL-lactide + CL 1     0.6KLCL lactide 2; caprolactone (CL) 18,500 caprolactone   2.5 18.5 KCL18,500 — — 18.5 KCO

EXAMPLE 1 Synthesis of Photopolymerized Biodegradable Hydrogels

PEG-Based Hydrogels

PEG-based biodegradable hydrogels are formed by the rapid laser or UVphotopolymerization of water soluble macromers. Macroners, in turn, aresynthesized by adding glycolic acid oligomers to the end groups of PEGand then capping with acrylic end groups. The PEG portions of themacromers confer water solubility properties, and subsequentpolymerization results in cell-nonadhesive hydrogels. Glycolic acidoligomers serve as the hydrolyzable fraction of the polymer network,while acrylic end groups facilitate rapid polymerization and gelation ofthe macromers.

In preparation for synthesis, glycolide (DuPont) or DL-lactide (Aldrich)was freshly recrystallized from ethyl acetate. PEG oligomers of variousmolecular weight (Fluka or Polysciences) were dried under vacuum at 110°C. prior to use. Acryloyl chloride (Aldrich) was used as received. Allother chemicals were of reagent grade and used without furtherpurification.

Macromer Synthesis

A 250 ml round bottom flask was flame dried under repeated cycles ofvacuum and dry argon. 20 gm of PEG (molecular weight 10,000), 150 ml ofxylene and 10 μm of stannous octoate were charged into the flask. Theflask was heated to 60° C. under argon to dissolve the PEG and cooled toroom temperature. 1.16 gm of glycolide was added to the flask and thereaction mixture was refluxed for 16 hr. The copolymer was separated oncooling and was recovered by filtration. This copolymer was separated oncooling and recovered by filtration. This copolymer (10 K PEG-glycolide)was used directly for subsequent reactions Other polymers were similarlysynthesized using DL-lactide or ε-caprolactone in place of glycolide andusing PEG of different molecular weights.

Synthesis of Photosensitive Oligomers (Macromers)

19 gm of 10 K PEG-glycolide copolymer was dissolved in 150 ml methylenechloride and refluxed with 1 ml acryloyl chloride and 1.2 ml oftriethylamine for 12 hr under an argon atmosphere. The solidtriethylamine hydrochloride was separated by filtration and the polymerwas precipitated by adding the filtrate to a large excess of hexane. Thepolymer (capped by an acrylate at both ends) was further purified byrepeated dissolution and precipitation in methylene chloride and hexanerespectively.

Table 2 lists certain macromers synthesized. The degree ofpolymerization of the glycolide chain extender was kept low so that allpolymers have approximately 10 ester groups per chain, or about 5 perchain end. When these polymers are photopolymerized, a crosslinkedthree-dimensional network is obtained. However, each chain segment inthe resulting network needs just one ester bond cleaved at either end to“degrade.” These ester cleavages enable the chain to dissolve in thesurrounding physiological fluid and thereby be removed from the implantsite. The resulting hydrolysis products, PEG and glycolic acid, arewater soluble and have very low toxicity.

TABLE 2 Macromers Synthesized Mol. Wt. % % ε- Calculated of CentralGlycolide Caprolactone Mol. Wt. of PEG Chain in in Extremities PolymerCode (daltons) Extremeties Extremities (daltons) Appearance 0.4 K  400100 — 580 Viscous liquid 1 KG  1000 100 — 300 Viscous liquid 4 KG  4000100 — 232 White solid 10 KG 10000 100 — 580 White solid 18.5 KG 18500100 — 1160  Yellow solid col8.5 KGCL 18500  50 — 580 White solid

Due to the presence of only a few units of glycolic acid per oligomericchain, the solubility properties of the photocrosslinkable prepolymersare principally determined by the central PEG chain. Solubility of themacromers in water and methylene chloride, both of which are solventsfor PEG, is not adversely affected as long as the central PEG segmenthas a molecular weight of 1,000 daltons or more. Solubility data for theprepolymers synthesized is given in Table 3.

TABLE 3 SOLUBILITY DATA Solvent 1 KG 4 KG 10 KG 18.5 KG TMP* DMSO — ▪ —▪ ▪ Acetone — ▪ ▪ ▪ — Methanol — ▪ — ▪ — Water — — — — ▪ Hexane ▪ ▪ ▪ ▪▪ Methylene — — — — — Chloride Cold Xylene ▪ ▪ ▪ ▪ — Hot Xylene — — — —— Benzene ▪ ▪ ▪ ▪ — —Soluble ▪Not Soluble *Trimethylolpropane glycolidetriacrylate

PEG chains with different degrees of polymerization of DL-lactide weresynthesized to determine the degree of substitution for which watersolubility of the macromers can be retained. The results are shown inTable 4. Beyond about 20% substitution of the hydrophilic PEG chain withhydrophobic DL-lactoyl or acrylate terminals leads to the macromersbecoming insoluble in water, though they still soluble in organicsolvents such as methylene chloride.

TABLE 4 Solubility of Macromers D.P.* of D.P.* of % extension Ethylenelactide or of PEG Solubility Oxide glycolide chain in water 420  4 0.1soluble 420 10 2.4 soluble 420 20 4.8 soluble 420 40 9.5 soluble 420 8019 insoluble  23  2 8.7 soluble  23  4 17.4 soluble  23 10 43.5insoluble  23 40 174 insoluble  5  4 80 insoluble  10  4 40 soluble*degree of polymerization

Photopolymerization

The macromers can be gelled by photopolymerization using free radicalinitiators, with the presence of two acrylic double bonds per chainleading to rapid gelation. A 23% w/w solution of various degradablepolymers in HEPES buffered saline containing 3 μl of initiator solution(300 mg/ml of 2,2-dimethoxy-2-phenyl-acetophenone in n-vinylpyrrolidone) was used. 100 μl of the solution was placed on a glasscoverslip and irradiated with a low intensity long wavelength UV (LWUV)lamp (Blak-Ray, model 3-100A with flood). The times required forgelation to occur were noted and are given below. These times aretypically in the range of 10 seconds. This is very significant becausethese reactions are carried out in air (UV initiatedphotopolymerizations are slow in air as compared to an inert atmosphere)and using a portable, low powered long wave UV (LWUV) emitting source.Oxygen, which often inhibits free radical reactions by forming specieswhich inhibit propagation, did not seem to slow down the polymerization.Such fast polymerizations are particularly useful in applicationsrequiring in situ gelations. This rapid gelation is believed to be dueto the formation of micelle-like structures between the relativelyhydrophobic polymerizable groups on the macromer, thereby increasing thelocal concentration of the polymerizable species in aqueous solution andincreasing polymerization rates.

Visible laser light is also useful for polymerization. Low intensity andshort exposure times make visible laser light virtually harmless toliving cells since the radiation is not strongly absorbed in the absenceof the proper chromophore. Laser light can also be transported usingfiber optics and can be focused to a very small area. Such light can beused for rapid polymerization in highly localized regions; gelaciontimes for selected prepolymers are given in Table 5. In each case, 0.2ml of a 23% w/v photosensitive oligomer solution is mixed with ethyleosin (10⁻⁴ M) and triethanol amine (0.01 to 0.1 M) and the solution isirradiated with an argon ion laser (American argon ion laser model 905emitting at 514 nm) at a power of 0.2-0.5 W/cm². The beam is expanded toa diameter of 3 mm and the sample is slowly scanned until gelationoccurs.

TABLE 5 Gelation Times UV polymerization* gelation time LaserPolymerization** Polymer (mean ± S.D.) (s) gelation time (s)  1 KG  5.3± 4.1 <1  4 KG 14.7 ± 0.5 <1  6 KG  9.3 ± 0.5 <1 10 KG  18. ± 0.8 <1 10KL  7.7 ± 0.5 <1 18 KG 23.3 ± 1.2 <1 20 KG 13.3 ± 0.5 <1 *Initiator:2,2-dimethoxy-2-phenylacetophenone, concentration 900 ppm: 0.2 ml of 23%monomer solution in PBS **Argon ion laser emitting at 514 nm. power 3W/cm²: ethyloeosin, triethanol amine initiating system: 0.2 ml of 23%monomer solution in PBS

Biodegradability

Biodegradation of the resulting polymer network is an important criteriain many biomedical applications. Degradation of poly(glycolic acid andpoly(DL-lactic acid) has been well documented in the literature. Thedegradation mainly takes place through the hydrolysis of the ester bond;the reaction is second order and highly pH dependent. The rate constantat pH 10 is 7 times faster than that at pH 7.2.

Such facile biodegradation is surprising becausepoly(α-hydroxyacidesters) are hydrophobic and highly insoluble in water.Accessibility of the polymer matrix to the aqueous surrounding istherefore limited. However, because the networks are hydrogels which areswollen with water, all the ester linkages in the network are inconstant contact with water with the aqueous surroundings. This resultsin a uniform bulk degradation rather than a surface degradation of thesegels.

Table 6 gives hydrolysis data for some of these networks; times listedare for complete dissolution of 60 mg of gel at pH 7.2 and 9.6. Asnoted, most of the gels dissolve within 12 hours at pH 9.6. 18.5 k geldissolves within 2.5 hr at pH 9.6 whereas 18.5 KCO gel does not dissolvein 3 days, indicating that the lactoyl, glycoloyl, or ε-caprolactoylester moiety is responsible for degradation of these networks. It alsocan be seen that the 18.5 KG gel hydrolyzes more rapidly than the 4 KGgel. This may be due to the reduced hydrophilicity and higher crosslinkdensity of the latter gel.

TABLE 6 Hydrolysis Data Oligomer Time taken to Time taken to used fordissolve gel at dissolve gel at gelation pH 9.6 (h) pH 7.2 (days)   4 KG62 5.5   10 KG 12.25 5.5 18.5 KG 2.25 >7 18.5 KCL >5 days >7 18.5 KCO >5days >7

Characterization of Macromers

FTIR spectra of the prepolymers were recorded on a DIGILAB model FTS15/90. The absorption at 1110 cm⁻¹ (characteristic C-0-C absorption ofPEG) shows the presence of PEG segments. The strong 1760 cm⁻¹ absorptionshows the presence of glycolic ester. The absence of hydroxyl groupabsorption around 3400 cm⁻¹ and a weak acrylic double bond absorption at1590 cm⁻¹ shows the presence of acrylic double bonds at the end groups.

500 MHz proton and 125 MHz carbon-13 spectra were recorded on a GE 500instrument The presence of a very strong peak at 4.9 ppm due to CH₂methylene from the PEG segment, a peak at 5.09 ppm due to the glycolicester segment and an acrylic proton singlet at 5.8 ppm can be easilyseen from proton NMR. The estimated molecular weight of PEG segment andglycolic acid segment for different copolymers is shown in Table 2. Thecarbonyl peak at 169.39 ppm from glycolic acid and 36.5 ppm peak frommethylene carbons from PEG in carbon-13 NMR are consistent with thereported chemical composition of these copolymers.

Differential scanning calorimetry (Perkin Elmer DSC-7) was used tocharacterize the oligomers for thermal transitions. The oligomers wereheated from −40° C. to 200° C. at a rate of 20° C./min, presumablycausing polymerization. The polymer was then cooled to −40° C. at a rateof 60° C./min and again heated to 200° C. at a rate of 20° C./min. Thefirst scans of biodegradable 18.5 K PEG glycolide tetraacrylate (18.5KG) oligomer were compared to that of the non-degradable 18.5 K PEGtetraacrylate (18.5 KCO) scan. It was seen that a glass transitionappears in the 18.5 KG at −2° C. while no such transition exists in the18.5 KCO. A small melting peak at 140° C. was also evident due to thefew glycolic acid mers which can crystallize to a limited extent. Themelting peak for PEG is shifted downwards in 18.5 KG to 57° C. from60.7° C. for 18.5 KCO. This is probably due to disturbance of the PEOcrystalline structure due to the presence of the glycolic acid linkages.In the third cycle, by which time the oligomers have presumablypolymerized, the Tg and Tm transitions for the glycolide segments can nolonger be seen, indicating that a crosslinked network has formed and theglycolic acid segments are no longer capable of mobility.

The degree of polymerization (D.P.) of the degradable segments added tothe central water soluble PEG chain was determined in several casesusing ¹H NMR. The experimentally determined D.P. was seen to be in goodagreement with the calculated number, as shown by FIG. 1A. Thus, thering opening reaction initiated by the PEG hydroxyls proceeds tocompletion, giving quantitative yields.

Determination of Total Water, Free Water Bound Water

Solutions of various degradable macromers were made as described above.Gels in the shape of discs were made using a mold. 400 μl of solutionwas used for each disc. The solutions were irradiated for 2 minutes toensure thorough gelation. The disc shaped gels were removed and driedunder vacuum at 60° C. for 2 days. The discs were weighed (W1) and thenextracted repeatedly with chloroform for 1 day. The discs were driedagain and weighed (W2). The gel fraction was calculated as W2/W1. Thisdata appears in Table 7.

Subsequent to extraction, the discs were allowed to equilibrate with PBSfor 6 hours and weighed (W3 after excess water had been carefullyswabbed away). The total water content was calculated as (W3−W2)×10/W3.Differential scanning calorimetry (DSC) was used to determine theeamount of free water that was available in the gels. A scan rate of 20°C./min was used and the heat capacity for the endotherm for watermelting was measured (H1). The heat capacity of HBS was also measured(H2). The fraction of free water was calculated as H1/H2. The residualwater was assumed to be bound due to hydrogen bonding with the PEOsegments. The presence of free water in the gels was indicated. Thisfree water can be expected to help proteins and enzymes entrapped insuch gels in maintaining their native conformation and reducingdeactivation. Thus these gels would appear to be suited for controlledrelease of biological macromolecules. The data for gel water content issummarized in Table 7

TABLE 7 Hydrogel Water content Polymer % Free % Bound % Total % Gel CodeWater Water Water Content   1 KG 68.4 14   82.3 ± 2.6 61.3 ± 5.2    4 KG78.0  9.3 87.3 ± 1.8 56.3 ± 0.9    6 KG 74.8 13.4 88.1 ± 3.3 66.5 ± 2.35  10 KG 83.7 10.8 94.5 ± 0.5 54.3 ± 0.6    10 KL 82.0  9.7 91.7 ± 0.563.9 ± 3.7  18.5 KG 71.8 22.3 94.0 ± 0.4 47.0 ± 4.9    20 KG 79.8 14.894.5 ± 0.4 44.5 ± 4.8 

EXAMPLE 2 Use of Multifunctional Macromers

30 g of a tetrafunctional water soluble PEG (MW 18,500) (PEG 18.5 k) wasdried by dissolving the polymer in benzene and distilling off the waterbenzene azeotrope. In a glove bag, 20 g of PEG 18.5 k, 1.881 g ofglycolide and 15 mg of stannous octoate were charged into a 100 ml roundbottom flask. The flask was capped with a vacuum stopcock, placed into asilicone oil bath and connected to a vacuum line. The temperature of thebath was raised to 200° C. The reaction was carried out for 4 hours at200° C. and 2 hours at 160° C. The reaction mixture was cooled,dissolved in dichloromethane and the copolymer was precipitated bypouring into an excess of dry ethyl ether. It was redissolved in 200 mlof dichloromethane in a 500 ml round bottom flask cooled to 0° C. Tothis flask, 0.854 g of triethylamine and 0.514 ml of acryloyl chloridewere added under nitrogen atmosphere and the reaction mixture wasstirred for 12 h at 0° C. The triethyl amine hydrochloride was separatedby filtration and the copolymer was recovered from filtrate byprecipitating in diethyl ether The polymer was dried at 50° C. undervacuum for 1 day.

EXAMPLE 3 Synthesis of a Photosensitive Macromer Containing DL-Lactide

PEG (MW) 20,000) (PEG 20 k) was dried by dissolving in benzene anddistilling off the water benzene azeotrope. In a glove bag, 32.43 g ofPEG 20 k, 2.335 g of DL-lactide and 15 mg of stannous octoate werecharged into a 100 ml round bottom flask. The flask was capped with avacuum stopcock, placed into a silicone oil bath and connected to avacuum line. The temperature of the bath was raised to 200° C. Thereaction was carried out for 4 hours at 200° C. The reaction mixture wascooled, dissolved in dichloromethane and the copolymer was precipitatedby pouring into an excess of dry ethyl ether. It was redissolved in 200ml of dichloromethane in a 500 ml round bottom flask cooled to 0° C. Tothis flask, 0.854 g of triethylamine and 0.514 ml of acryloyl chloridewere added under nitrogen atmosphere and the reaction mixture wasstirred for 12 hours at 0° C. The triethyl amine hydrochloride wasseparated by filtration and the copolymer was recovered from filtrate byprecipitating in diethyl ether. The polymer was dried at 50° C. undervacuum for 1 day.

EXAMPLE 4 Synthesis of a Photosensitive Precursor Containing DL-Lactideand ε-Caprolactone

PEG (MW 600) (PEG 0.6 k) was dried by dissolving in benzene anddistilling off the water benzene azeotrope. In a glove bag, 0.973 g ofPEG 0.6 k, 0.467 g of DL-lactide along with 0.185 g of ε-caprolactoneand 15 mg of stannous octoate were charged into a 50 ml round bottomflask. The flask was capped with a vacuum stopcock, placed into asilicone oil bath and connected to a vacuum line. The temperature of thebath was raised to 200° C. The reaction was carried out for 4 hours at200° C. and 2 hours at 160° C. The reaction mixture was cooled,dissolved in dichloromethane and the copolymer was precipitated bypouring into an excess of dry ethyl ether. It was redissolved in 50 mlof dichloromethane in a 250 ml round bottom flask cooled to 0° C. tothis flask, 0.854 g of triethylamine and 0.514 ml of acryloyl chloridewere added under nitrogen atmosphere and the reaction mixture wasstirred for 12 hours at 0° C. The triethyl amine hydrochloride wasseparated by filtration and the copolymer was recovered from filtrate byprecipitating in diethyl ether. The polymer was dried at 50° C. undervacuum for 1 day and was a liquid at room temperature.

EXAMPLE 5 Selection of Dyes for Use in Photopolymerization

It is possible to initiate photopolymerization with a wide variety ofdyes as initiators and a number of electron donors as effectivecocatalysts. Table 8 illustrates photopolymerization initiated byseveral other dyes which have chromophores absorbing at widely differentwavelengths. All gelations were carried out using a 23% w/w solution of18.5 KG in HEPES buffered saline. These initiating systems comparefavorably with conventional thermal initiating systems, as can also beseen from Table 8. Other photoinitiators that may be particularly usefulare 2-methoxy-2-phenyl acetophenone and camphorquinone.

TABLE 8 Polymerization Initiation of 18.5 KG PEG TEMPER- GEL LIGHT ATURETIME INITIATOR SOURCE* ° C. (SEC) Eosin Y, 0.00015M; S1 with 25 10Triethanolamine 0.65M UV filter Eosin Y, 0.00015M; S4 25 0.1Triethanolamine 0.65M Methylene Blue, 0.00024M; S3 25 120p-toluenesulfinic acid, 0.0048M 2,2-dimethoxy-2-phenyl S2 25 8acetophenone 900 ppm Potassium persulfate — 75 180 0.0168M PotassiumPersulfate — 25 120 0.0168M; tetramethyl ethylene-diamine 0.039MTetramethyl ethylene- S1 with 25 300 diamine 0.039M; UV filterRiboflavin 0.00047M *LIST OF LIGHT SOURCES USED CODE SOURCE S1 Mercurylamp, LEITZ WETSLER Type 307-148.002, 100 W S2 Black Ray longwave UVlamp, model B-100A W/FLOOD S3 MELLES GROIT He—Ne laser, 10 mW output, 1= 632 nm S4 American laser corporation, argon ion laser, model909BP-15-01001; λ = 488 and 514 nm

Numerous other dyes can be used for photopolymerization. These dyesinclude but are not limited to: Erythrosin, phloxine, rose bengal,thioneine, camphorquinone, ethyl eosin, eosin, methylene blue, andriboflavin. The several possible cocatalysts that can be used includebut are not limited to: N-methyl diethanolamine, N,N-dimethylbenzylamine, triethanol amine, triethylamine, dibenzyl amine, N-benzylethanolamine, N-isopropyl benzylamine, and N-vinyl pyrrolidinone.

EXAMPLE 6 Thermosensitive Biodegradable Gels from N-Isopropyl AcrylamideSynthesis of Low Molecular Weight Polyisopropyl Acrylamide

N-isopropyl acrylamide (NIPAAm) was recrystallized from 65:35 hexanebenzene mixture. Azobisisobutyronitrile (AIBN) was recrystallized frommethanol. 1.5 g of NIPAAm was polymerized using 3 mg of AIBN and 150 mgof mercaptoethanol in 1:1 acetone water mixture (24 hours at 65° C.).The viscous liquid after polymerization was purified by dissolving inacetone and precipitating in diethyl ether. Yield 80%.

This hydroxy terminated low molecular weight poly(NIPAAm) was used inchain extension reactions using glycolide and subsequent endcappingreaction using acryloyl chloride as described in other examples.

1 g of modified poly(NIPAAm) based oligomer and 0.2 g 1 KL weredissolved in water at 0° C. and polymerized at 0° C. using2-2-dimethoxy-2-phenylacetophenone (900 PPM).

EXAMPLE 7 In Vitro Degradation

The gels were extracted as described in Example 1 to remove theunpolymerized macromer fraction fraction and the gels were then placedin 50 mM HEPES buffered saline (0.9% NaCl), pH 7.4 at 37° C. Duplicatesamples were periodically removed, washed with fresh HBS and dried at100° C. for 1 day and weighed to determine mass loss in the gel. Thecompositions of the various gels used were the same as described in theprevious examples. Table 9 shows the extent of degradation of these gelsgiven as percent of mass lost over time. The respective times are givenin parenthesis along with the mass loss data.

TABLE 9 Gel Degradation  1 KG 20.1% (1 d), 20.36 ± 0.6 (2 d), 21.7 ± (6d), 28.8 ± 16.6 (10 d) estimated total Degradation time 45 days.  4 KG38.9 (1 d), 60.3 ± 4.2 (2 d), 78.9 (3 d), 99.3 ± 4.7 (6 d). Totaldegradation time 5.5 days.  6 KG 18.3 ± 6.8 (1 d), 27.4 ± 1.0 (2 d),32.8 ± 11.3 (3 d), 104.8 ± 3.2 (5 d). total degradation time 4.5 days 10KG 0.6 ± 0.6 (8 hr), 100 (1 d). Total degradation time 1 day. 10 KL 10.0± 4.84 (2 d), 6.8 ± 1.7 (3 d), 4.5 ± 3.1 (6 d), 8.0 ± 0.2 (10 d). Totaldegradation time estimated to be 20 days. 20 KG 68.1 ± 4.2 (8 hr), 99.7± 0.3 (1 d). Total degradation time 15 hr.

EXAMPLE 8 Fibroblast Adhesion and Spreading

The in vitro response of Human foreskin fibroblast (HFF) cells tophotopolymerized gels was evaluated through cell culture on polymernetworks. 0.2 ml of monomer solution was UV polymerized on an 18×18 mmglass coverslips under sterile conditions. HFF cells were seeded onthese gels at a cell density of 1.8×10⁴ cells/sq cm of coverslip area inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetalcalf serum. The gels were incubated for 6 hr at 37° C. in a 5% CO₂environment, at the end of which they were washed twice with phosphatebuffered saline (PBS). The adherent cells were fixed using a 2%glutaraldehyde solution in PBS. The gels were examined under a phasecontrast microscope at a magnification of 200×, and the number ofadherent and spread cells evaluated by examining five fields selected atpredetermined locations on the coverslips.

The number of adherent cells is reported in Table 10 along with thosefor glass control surfaces. Cell adhesion is seen to be dramaticallylowered on gel-coated glass.

TABLE 10 Cell Adhesion Surface Attached Cells/cm² glass 13220 ± 3730 18.5 KG 250 ± 240 18.5 KCL 1170 ± 1020 18.5 KCO 390 ± 150

It can be easily seen from Table 10 that these gels are highly resistantto cellular growth. Even the 18.5 KCL is still less than 10% of theglass. Cells attached to the glass surface show a flattened andwell-spread morphology whereas the few cells that are attached to thegel are rounded and loosely attached. This may result from the fact thathydrated PEG chains have a high motility and have been shown to beeffective in minimizing protein adsorption. One of the mechanisms bywhich cell adhesion is mediated is through the interaction of cellsurface receptors with adsorbed cell adhesion proteins. Thus thereduction in overall protein adsorption results in minimal cell adhesionprotein adsorption and reduced cell adhesion.

EXAMPLE 9 Release of Protein (Bovine Serum Albumin) from Polymers

1 KG was used for this study. This macromer was liquid at roomtemperature and was used as such. 1 mg of bovine serum albumin (BSA) wasadded per ml of monomer solution along with 0.9 mg/ml of2,2-dimethoxy-2-phenyl-acetophenone as initiator. The protein wasdissolved in the monomer solution and disc shaped gels were made byexposing 0.2 g of macromer mixture to LWUV for 1 min. Two such discswere placed in a flask containing 20 ml of PBS and incubated at 37° C.Two aliquots of 20 μl each were removed from these flasks periodicallyand the amount of BSA released was assayed using the Bio-Rad totalprotein assay. The release profile for BSA is shown in FIG. 3A. It canbe seen that the release of BSA is relatively steady over more than amonth.

EXAMPLE 10 Enzyme Release Assay

Water solubility of the macromers means gelation can be carried out in anon-toxic environment. This makes these materials suitable forintraoperative uses where in situ gelation is needed. Since theprecursors are water soluble, the gels can be used as drug deliveryvehicles for water soluble drugs, especially macromolecular drugs suchas enzymes, which would otherwise be denatured and lose their activity.Release of lysosome and tPA from the polymers was used to illustrate thefeasibility of using biodegradable hydrogels for controlled release ofbiomolecules.

Lysozyme release

The enzyme lysozyme (MW:14,400) is a convenient model for release of alow molecular weight protein from a biodegradable gel. The Biorad totalprotein assay was used to quantify the enzyme released. The enzyme wasdissolved in PBS at a concentration of 20 mg/ml. The monomerPEG-dl-lactic acid-diacrylate was dissolved in PBS to produce a 40%solution. The lysozyme solution was added to the monomer solution toattain a 24% monomer solution. The monomer/lysozyme solution waspolymerized under UV in a cylindrical mold, using 30 μl of the initiator2,2-dimethoxy-2-phenyl-acetophenone in 1-vinyl-2-pyrrolidone (30 mg/ml)as the initiator. The polymer was cut into 10 equal sized pieces andimmersed in 10 ml PBS. Samples of the PBS were withdrawn at intervalsand assayed for lysozyme released into the PBS. Lysozyme was releasedfrom the PEG-DL-lactic acid-diacrylate gel over an 8 day interval, withthe maximum rate of release occurring within the first 2 days, as shownby FIG. 3B.

Release of recombinant t-PA

Three macromers were used for these studies: 1 KL, 4 KG, and 18.5 KG.The 1 KL macromer was liquid at room temperature and was used as such.The second macromer, 4 KG, was used as a 75% w/w solution in PBS. Thethird composition was a mixture of equal parts of 1 KL and a 50% w/wsolution of 18.5 KG. 3.37 mg of tissue plasminogen activator (singlechain, recombinant, M.W. 71,000) was added per gram of macromer solutionalong with 0.9 mg/ml of 2,2 dimethoxy 2 phenyl acetophenone asinitiator. The protein was dissolved with the macromer and disc shapedgels were made by exposing 0.2 g of macromer mixture to LWUV for 1minute. Two such discs were rinsed with PBS, placed in a flaskcontaining 5 ml of PBS and incubated at 37° C. Two aliquots of 100 μleach were removed from these flasks periodically and the amount ofactive t-PA released was assayed using a chromogenic substrate assay(Kabi-vitrum). The release profiles from the 1 K lactide gels, 4 Kglycolide gels, and the 50/50 1 K glycolide/18.5 K glycolide are shownin FIGS. 4A-4C. Fully active tPA can be released for periods up to atleast two months.

By selecting an appropriate formulation, the release rate can betailored for a particular application. It is also possible to combineformulations with different molecular weights so as to synergisticallyachieve appropriate attributes in release and mechanicalcharacteristics.

For prevention of postoperative adhesions, in addition to the barriereffect of the gels, the gels can be loaded with a fibrinolytic agent tolyse incipient filmy adhesions which escape the barrier effect. Thisfurther enhances the efficacy of biodegradable gels in adhesionprevention.

EXAMPLE 11 Toxicity of Polymers and Commercial Adhesives

To evaluate the toxicity of in situ polymerization of the macromersolutions described herein, as compared to commercial adhesives, 100 μlof 18.5 KCO prepolymer solution was placed on the right lobe of a ratliver and gelled by exposing it to LWUV for 15 sec; similarly, a fewdrops of a n-butyl cyanoacrylate based glue were placed on the leftlobe. The liver was excised after a week, fixed in 10% neutral bufferedformalin, blocked in paraffin, sectioned and stained using hematoxylinand eosin.

No adverse tissue reaction was evident on the surface of the lobeexposed to the biodegradable gel. No inflammatory reaction to thepolymerization process can be seen. The epithelium looks normal, with noforeign body reaction.

In comparison, the lobe exposed to cyanoacrylate glue shows extensivetissue necrosis and scarring with 10-30 cell deep necrotic tissue.Fibrosis is evident in the necrotic portions close to underlying normaltissue.

EXAMPLE 12 Prevention of Post-Surgical Adhesions with PhotopolymerizedBiodegradable Polymer

A viscous sterile 23% solution in phosphate buffered saline (8.0 g/lNaCl, 0.201 g/l KCl, 0.611 g/l Na₂HPO₄, 0.191 g/l KH₂PO₄, pH 7.4) ofpolyethylene glycol (M.W. 18,500) which has been chain extended on bothends with a short polyglycolide repeat unit (average number ofglycolidyl residues: 10 on each end) and which has been subsequentlyterminated with an acrylate group was prepared. Initiator needed for thecrosslinking reaction, 2,2-dimethoxy-2-phenyl acetophenone, was added tothe macromer solution to achieve an initiator concentration of 900 ppm.A 30 second exposure to a long wave UV lamp (Blak Ray) is sufficient tocause polymerization

Animal models evaluated

Animal models evaluated included a rat cecum model and a rabbit uterinehorm model In the rat cecum mode, 6 out of 7 animals treated with themacromer solution showed no adhesions whatsoever, while untreatedanimals showed consistent dense adhesion formation. In the rabbituterine horn model, a significant (p<0.01) reduction in adhesionformation was seen in the animals treated with the gel. Studiesconducted in rats using only the ungelled viscous precursor solution (noLWUV) failed to prevent the formation of adhesions.

Rat cecum model

Twenty-one Sprague Dawley male rats having an average weight of 250 gmwere divided into three groups for treatment and two for controls. Theabdomen was shaved and prepared with a betadine solution. A midlineincision was made under Equithesin anesthesia. The cecum was located and4 to 5 scrapes were made on a region about 2×1 cm on one side of thececum, using a 4×4 in gauze pad to produce serosal injury and punctatebleeding. The abdominal incisions in these animals were closed using acontinuous 4-0 silk suture for the musculoperitoneal layer and 7.5 mmstainless steel staples for the cutaneous layer. A topical antibioticwas applied at the incision site.

The first group consisted of 7 animals serving as controls withouttreatment, to confirm the validity of the model. The second group servedas a control with the application of the precursor but withoutphotopolymerization to form the hydrogel. After induction of the cecalinjury, about 0.25 ml of the precursor solution was applied to theinjury site using a pipet. The abdominal incision was then closed asabove.

The third group served as the gel treatment group and was prepared asthe second group except that the precursor film was exposed to a LWUVlamp for 45 seconds to cause gelation. Both the obverse and reversesides of the cecum were similarly treated with precursor and light. Noattempt was made to dry the surface of the tissue, to remove blood, orto irrigate the area prior to treatment.

The animals were sacrificed at the end of two weeks by CO₂ asphyxiation.The incisions were reopened and adhesions were scored for location,extent, and tenacity. The extent of adhesions was reported as apercentage of the traumatized area of the cecum which forms adhesionswith adnexal organs or the peritoneal wall. Tenacity of the adhesionswas scored on a scale from 0 to 4: no adhesions—grade 0; tentativetransparent adhesions which frequently separate on their own—grade 1;adhesions that give some resistance but can be separated by hand—grade2; adhesions that require blunt instrument dissection to separate—grade3; and dense thick adhesions which require sharp instrument dissectionin the plane of the adhesion to separate—grade 4.

Rat cecum model results

The control group without treatment shows consistently dense andextensive adhesions. The extent of abraded area covered with adhesionswas seen to be 73±21% (mean±S.D., n=7). The severity of adhesions wasgrade 3.5±0.4. Most of the adhesions were dense and fibrous, involvingthe cecum with itself, with the peritoneal wall and with other organssuch as the liver, small intestine, and large intestine. Frequently thenesentery was seen to be involved in adhesions. In the control groupwith the application of precursor solution but without gelation byexposure to the LWUV lamp, the extent of adhesion was 60±24% (n=7), andthe severity of adhesions was 3.1±0.4. In the gel treated group, thececum was seen to be completely free of adhesions in 6 out of 7 animals.In one case, a grade 2 adhesion was seen with the mesentery over 10% ofthe area and a grade 2.5 adhesion was seen over 15% of the area,bridging the cecum to the sutures on the site of the incision in theperitoneal wall. The overall adhesion extent for the group was 4%, andthe overall severity was 0.32. No evidence of residual gel was visible,the gel presumably having degraded within the prior two weeks. The cecumappeared whitish with a fibrous layer on the surface in the controlgroup, but the tissue appeared healthy and normal in animals treatedwith the gel.

Rabbit uterine horn model

Eight sexually mature female New Zealand rabbits between 2 and 3 kg inweight were prepared for surgery. A midline incision was made in thelower abdominal region under Rompun, Ketamine, and Acepromazineanesthesia. The uterine horns were located and the vasculature to bothhorns was systematically cauterized to induce an ischemic injury. Oneanimal was rejected from the study due to immature uterine horns. Sevenrabbits were selected for the treatment with only the photopolymerizablehydrogel and two animals were selected for evaluating the combinedefficacy of the hydrogel with a fibrinolytic agent, tissue plasminogenactivator (tPA). 5 mg of tPA/ml macromer solution was used in the lattercase. After cauterization, macromer solutions (0.5 ml) were appliedalong the horn and allowed to coat the surface where the cauterizationinjury had been induced. After uniform application of the solution wascomplete, the horns were exposed to a LWUV lamp for 1 min to inducegelation. The procedure was repeated on the reverse side of the horns.The incisions were then closed using a continuous 2-0 Vicryl (Ethicon)suture for the musculoperitoneal layer and a 0 Vicryl (Ethicon) suturefor the cutaneous layer. No prophylactic antibiotics were administered.No postoperative complications or infections were observed. Five animalswere used in the control group. The ischemic injury was made asdescribed and the incision was closed without the application of theprecursor; all techniques were identical between the treatment group andthe control group.

Controls were used where the same animal model was subjected to surgerywithout application of the macromer; all surgical techniques wereidentical between the treatment group and the historical controls.

The rabbits were reoperated under Ketamine anesthesia at the end of twoweeks to evaluate adhesion formation; they were sacrificed byintrocardiac KCl injection. Adhesion formation was evaluated for extentand tenacity. Extent of adhesion formation was evaluated by measuringthe length of the uterine horn that formed adhesions with itself or withthe peritoneal wall or other organs. Tenacity of adhesion was classifiedas either filmy or fibrous. Filmy adhesions were usually transparent,less strong, and could be freed by hand. The fibrous adhesions weredense, whitish, and usually required sharp instrument dissection to befreed. In cases where only a single filmy adhesion band was evident, ascore of 5%. was assigned

Typical samples of the horn were excised for histology and were fixed ina 10%. neutral buffered formalin solution. Paraffin sections of thesamples were stained using hematoxylin and eosin.

Rabbit uterine horn model results

The adhesion score is the % of affected area occupied by the adhesions,with grading of each as being filmy or fibrous. Distorted horn anatomieswere observed in control animals. The mean score in the control groupwas 50 ±15% of the affected area of the horn being occupied by adhesionswith 10% of these being filmy and 90% fibrous. Distorted horn anatomieswere observed. The uterine horn of an animal used as a control; showedadhesions over 66% of the horn surface. The group of animals treatedonly with the photopolymerized macromer showed an adhesion score of13±11.4% (n=10). Of these, 4 animals showed less than 5% adhesions withonly an occasional filmy band visible.

The animals treated with photopolymerized gel containing tPA showedfurther improved results over the “gel only” animals. One animals showeda filmy band on both the right and left horn. They were assigned a scoreof 5% with a total score of 10%. The other animal did not show anyadhesions at all. Thus the total score for these animals was 5±5%.

Normal horn anatomy in a typical horn which has undergone gel treatmentadhesions are filmy in all cases and no dense bands are seen. No tracesof the remaining gel could be observed. Typical samples of horns showingfilmy adhesions showed some fibrous tissue with a 6-15 cell thick layerof fibroblasts showing some collagen fibrils but no formation of densecollagen fibers. The horns showing no adhesions occasionally showed a1-4 cell thick layer of fibroblasts, but mostly a normal epithelium withno evidence of inflammatory cells.

This same procedure was slightly modified as described below as a bettermode of using the polymers to prevent postoperative adhesions using therat uterine horn model.

Female rats were anesthetized with pentobarbital (50 mg/kg,intraperitoneally), and a midline laparotomy was performed. The uterinehorns were exposed, and the vasculature in the arcade feeding the hornswas systematically cauterized using bipolar cautery; the most proximaland most distal large vessel on each horn were not cauterized. Followingthis, the antimesenteric surface of each horn was cauterized at two 1 mmdiameter spots on each horn, each separated by a 2 cm distance, the paircentered along the length of each horn. Following injury, 0.5 ml ofmacromer solution was applied per horn and was gelled by exposure tolong wavelength ultraviolet light (365 nm, approximately 20 mW/cm²) for15 sec per surface on the front side and on the back side each. Theuterus was replaced in the peritoneal cavity, and the musculoperitonealand skin layers were closed.

The macromer consisted of a PEG chain of MW 8,000 daltons, extended onboth sides with a lactic acid oligomer of an average degree ofpolymerization of 5 lactidyl groups, and further acrylated nominally atboth ends by reaction with acryloyl chloride. In one batch, Batch A, thedegree of acrylation was determined by NMR to be approximately 75%, andin another, Batch B, it was determined to be greater than approximately95%. The macromer was dissolved in saline at a specified concentration,and the initiation system used was 2,2-dimethoxy-2-phenyl acetophenonefrom a stock solution in N-vinyl pyrrolidinone, the final concentrationof 2,2-dimethoxy-2-phenyl acecophenone being 900 ppm and the finalconcentration of N-vinyl pyrrolidinone being 0.15%.

In one set of experiments, macromer from Batch A was applied in varyingconcentrations, and adhesions were scored at 7 days postoperatively.Scoring was performed by two means. The length of the horns involved inadhesions was measured with a ruler, and the fraction of the totallength was calculated. The nature of the adhesions was also scored on asubjective scale, 0 being no adhesions, 1 being filmy adhesions that areeasily separated by hand, and 2 being dense adhesions that can only beseparated by sharp instrument dissection. Furthermore, one of thesamples contained tissue-plasminogen activator (t-PA), which is known toreduce adhesions, at a concentration of 0.5 mg/ml (0.5%) macromersolution. The results are shown in Table 11 for macromer batch A andbatch B.

In a third set of experiments, adhesions were formed in female rats asdescribed above, and the adhesions were surgically lysed 7 days afterthe initial surgery. The extent and grade of adhesions was scored duringlysis. The animals were divided into two groups, and one group wastreated with macromer from Batch B at a concentration of 10%. Theresults are shown in Table 11 as batch B, 10%.

TABLE 11 Reduction of Adhesions with Polymer. Extent of Grade ofConcentration adhesions adhesions Number of macromer % (S.D.) (0-2)Animals Polymer A 15% 24.6 (3.1)  1.1 (0.1) 7 20% 33.6 (9.8)  1.2 (0.3)7 25% 37.5 (11.1) 1.2 (0.1) 7 30% 54.2 (12.0) 1.6 (0.4) 6 20% + t-PA.18.3 (6.4)  1.1 (0.1) 6 Control (saline) 72.6 (18.7) 1.5 (0.2) 7 PolymerB  5% 22.1 (4.2)  1.2 (0.1) 7 10% 10.0 (5.1)  1.0 (0)   7 15% 17.8(5.7)  1.0 (0)   7 20% 26.3 (11.4) 1.4 (0.2) 7 Control (saline) 75.9(4.4)  1.8 (0.3) 7 Polymer B. 10% Scoring group performed that at:became: time of Controls 85.9 (9.7)  1.8 (0.1) 7 lysis Time of Treatment79.4 (6.8)  1.7 (0.2) 7 lysis 7 days Controls 78.8 (11.3) 1.8 (0.1) 7post-lysis 7 days Treatment 28.2 (5.1)  1.0 (0)   7 post-lysis

The above results illustrate that the photopolymerized macromer canreduce or prevent post operative adhesions in both primary adhesions andadhesiolysis models, and moreover that the gel can be used to locallyrelease a drug to exert a combined beneficial effect.

EXAMPLE 13 Nerve Anastomosis

The sciatic nerve of a rat was aseptically severed using a scalpel andallowed to pull apart. The two ends of the nerve were reopposed usingsterile forceps, and a 50 solution in buffer of polymer 1 KL, a macromermade from PEG 1 K with lactide chain extension and acrylate termination,with 0.1% 2,2-dimethoxy-2-phenoxy acetophenone was applied to the nervestumps. The affected area was illuminated with a 100 W LWUV lamp for 60seconds, and an adhesive bond was observed to form between the proximaland distal nerve stumps.

To ensure the biocompatibility of the applied material with the nervetissue, the same solution of macromer was applied to nonsevered ratsciatic nerves, and the area of the incision was closed using standardsmall animal surgical technique. The area was reopened at 1 hour or 24hour postoperatively, and the affected area of the nerve was removed enblock and prepared for transmission electron microscopy. Nomorphological differences were observable between the treated nerves ateither time point as compared to control rat sciatic nerves that wereotherwise nonmanipulated, even though they had been traumatized andmanipulated.

EXAMPLE 14 Evaluation of PEG Based Degradable Gels as Tissue Adhesives

Abdominal muscle flaps from female New Zealand white rabbits wereexcised and cut into strips 1 cm×5 cm. The flaps were approximately 0.5to 0.8 cm thick. A lap joint, 1 cm×1 cm, was made using two such flaps.Two different compositions, 0.6 KL and 1 KL, were evaluated on thesetissues. Both these compositions were viscous liquids and were usedwithout further dilution. 125 μl of ethyl eosin solution in N-vinylpyrrolidone (20 mg/ml) along with 50 μl of triethanolamine was added toeach ml of the adhesive solution 100 μl of adhesive solution was appliedto each of the overlapping flaps. The lap joint was then irradiated byscanning with a 2 W argon ion laser for 30 sec from each side. Thestrength of the resulting joints was evaluated by measuring the forcerequired to shear the lap joint. One end of the lap joint was clampedand an increasing load was applied to the other end, while holding thejoint was clamped and an increasing load was applied to the other end,while holding the joint horizontally until it failed. Four joints weretested for each composition. The 1 KL joints had a strength of 6.6±1.0KPa (mean ±S.D.), while the 0.6 KL joints had a strength of 11.4±2.9KPa. It is significant to note that it was possible to achievephotopolymerization and reasonable joint strength despite the 6-8 mmthickness of tissue. A spectrophotometric estimate using 514 nm lightshowed less than 1% transmission through such muscle tissue.

EXAMPLE 15 Coupling of Photopolymerizable Groups to Proteins (Albumin)

PEG (M.W. 2,000) monoacrylate (5 g) was dissolved in 20 mldichloromethane. Triethyl amine (0.523 g) and2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) (0.017 g) wereadded and the reaction was allowed to proceed for 3 hours at 0° C. undernitrogen atmosphere. The reaction mixture was then filtered and thedichloromethane evaporated to dryness. The residue was redissolved in asmall amount of dichloromethane and precipitated in diethyl ether. Thepolymer was then filtered and dried under vacuum for 10 hours and useddirectly in the subsequent reaction with albumin.

1 g of bovine serum albumin was dissolved in 200 ml of sodiumbicarbonate buffer at pH 9. Tresyl activated PEG monoacrylate (5 g) wasadded and the reaction was stirred for 24 hours at 25° C. Albumin wasseparated by pouring the reaction mixture into acetone. It was furtherpurified by dialysis using a 15,000 daltons cutoff dialysis membrane. A10% w/v solution of the PEG acrylated albumin could be photopolymerizedwith long wave UV radiation using 0.9 mg/ml of 2,2 dimethoxy 2phenylacetophenone as the initiator. In this gel the degradable segmentis the protein albumin.

EXAMPLE 16 Modification of Polysaccharides (Hyaluronic Acid)

In a dry 250 ml round bottom flask, 10 grams of PEG 400 monomethacrylatewas dissolved in 100 ml dry dioxane, to which 4.053 g of carbonyldiimidazole (CDI) was slowly introduced under nitrogen atmosphere andthe flask was heated to 50° C. for 6 h. Thereafter the solvent wasevaporated under vacuum and the CDI activated PEG monomer was purifiedby dissolving in dichloromethane and precipitating in ether twice.

1 g of hyaluronic acid, 5 g of CDI activated PEG 400 monoacrylate weredissolved in 200 ml sodium borate buffer (pH 8.5) and the solution wasstirred for 24 hours. It was then dialyzed using a 15,000 dalton cutoffdialysis membrane to remove unreacted PEG. A 10% w/v solution of theacrylated hyaluronic acid was photopolymerized with long wave UVradiation, using 0.9 mg/ml of 2,2-dimethoxy-2-phenylacetophenone as theinitiator. In this gel, the degradable region is hyaluronic acid.

EXAMPLE 17 PEG Chain Extended with Polyorthocarbonates and Capped withUrethane Methacrylate

3,9-bis(methylene) 2,4,8,10-tetraoxaspiro [5,5] undecane (1 g) andpolyethylene glycol (molecular weight, 1,000, 7.059 g) were weighed intoa 250 ml Schlenk tube under dry nitrogen atmosphere in a glove bag. 50ml of dry tetrahydrofuran was introduced under nitrogen atmosphere andreaction mixture was stirred for 6 hours at 50° C. This is a typicalstep growth reaction with a disturbed stoichiometry, resulting in lowmolecular weight poloyorthocarbonate with terminal hydroxy groups. Theoligomer was separated by precipitating in hexane and dried undervacuum. 5 g of cligomer was redissolved in dry THF to which 20 μl ofdibutyltindilaurate and 2 ml of 2-isocyanatoethyl methacrylate wereslowly introduced and temperature was raised to 50° C. It was held therefor 6 hours and cooled. The product was separated by precipitation inhexane. In this gel, the degradable region is a polyorthocarbonate.

EXAMPLE 18 Microencapsulation of Animal Cells

A 23% w/w solution of 18.5 KG in HEPES buffered saline (5 ml) was usedto resuspend 10⁶ CEM-SS cells. Ethyl eosin (10⁻⁴ M) was used as asolution in N-vinyl pyrrolidone as the initiator and triethanolamine(0.01 M) was used as the coinitiator. The solution was then exposedthrough a coextrusion apparatus to an argon ion laser (514 nm, 2 Watts)The coextrusion apparatus had mineral oil as the fluid flowing annularly(flow rate 4 ml/min) around an extruding stream of the precursor cellsuspension (flow rate 0.5 ml/min). The microdriplets gelled rapidly onbeing exposed to the laser light and were collected in a containercontaining PBS. The oil separated from the aqueous phase and themicrospheres could be collected in the PBS below. The microspheresformed were thoroughly washed with PBS buffer to remove unreactedmonomer and residual initiator. The size and shape of microspheres wasdependent on extrusion rate and extruding capillary diameter (18 Ga to25 Ga). The polymerization times were dependent on initiatorconcentration (ethyl eosin 5 μM to 0.5 mm, vinyl pyrrolidone (0.001% to0.1%), and triethanolamine (5 mM to 0.1 M), laser power (120 mW to 2 W),and monomer concentration (>10% w/v). Spheres prepared using this methodhad a diameter from 500 μm to 1,200 μm. The polymerizations were carriedout at physiological pH in the presence of air. This is significantsince radical polymerizations may be affected by the presence of oxygen.Cell viability subsequent to encapsulation was checked by trypan blueexclusion assay and the encapsulated cells were found to be more than95% viable after encapsulation.

EXAMPLE 19 Various Formulations for the Prevention of Post OperativeAdhesions

The utility of PEG-oligo (α-hydroxy acid) diacrylates and tetraacrylatesto prevent postoperative adhesions was evaluated in the rabbit uterinehorn model as described above. The following polymers were synthesized,as described above: PEG 6 K lactide diacrylate (6 KL), PEG 10 K lactidediacrylate (10 KL). PEG 18.5 K lactide (18.5 KL), PEG 20 K lactide (20KL). Solutions with 24% polymer in PBS with 900 ppm2,2-dimethoxy-2-phenyl acetophenone, were prepared as described above.The solutions were applied to the uterine horn after cautery at thevascular arcade and illuminated with a 365 nm LWUV lamp, as describedabove. In one formulation, 18.5 KL, 5 mg t-PA was mixed into thesolution before application. Controls consisted of animals manipulatedand cauterized but not treated with macromer solution. Measurement wasperformed on the 14th±1 day. Extent of adhesion was estimated from thefraction of the horn that was involved in adhesions, and the tenacity ofadhesions was scored as 0, no adhesions; 1, filmy adhesions that offerno resistance to dissection; 2, fibrous adhesions that are dissectableby hand; 3, fibrous adhesions that are dissectable by blunt instruments;and 4, fibrous adhesions that are dissectable by sharp instruments. Theresults were as follows, where the extent of adhesions and the tenacityof the adhesions are shown.

TABLE 12 Efficacy of Polymer in Preventing Adhesions. Number of Extent,Tenacity, Formulation animals %, ± S.D. 0-4 ± S.D.   6 KL 7 0.9 ± 1.70.9 ± 0.7   10 KL 7 0 ± 0 0 ± 0   20 KL 6 4.4 ± 5.0 0.9 ± 0.7 18.5 KL 7 8.9 ± 13.1 1.6 ± 1.3 t-PA Control 7 35 ± 22 3.3 ± 0.6

EXAMPLE 20 Polymerization of Ultrathin Layers of Polymer on the Surfaceof Blood Vessels to Reduce Thrombosis After Vessel Injury

Blood vessels were harvested from rats and were rinsed free of blood.The endothelium of the vessel were removed by inserting a wooden doweland rotating the vessel over the dowel. One vessel was used as acontrol, and was exposed to flowing blood as described below withoutfurther modification. Another vessel was treated first by exposure toeosin Y at 1 mM in saline, then rinsed in HEPES buffered saline, thenfilled with a solution of PEG-MA, PEG 10 K with acrylate end-cappedoligomers of DL lactide, containing triethanolamine (TEA) (100 mM) andN-vinylpyrrolidone (VP) (0.15%) and then illuminated by exposure to anargon ion laser at 0.5 W/cm² for 15 sec. The nonpolymerized prepolymermixture in the lumen of the vessel was rinsed away with saline. Humanblood was collected from the antecubital vein and was anticoagulatedwith heparin at 2 units/ml. This blood was perfused through each vesselby a syringe pump at a flow rate corresponding to a wall shear rate ofapproximately 200/s for 7 min. The vessel was then superficially rinsedin saline and fixed in formaldehyde.

The treated vessel did not appear colored or different in color afterperfusion compared to its color before perfusion, while the untreatedcontrol vessel appeared blood red. Thin segments of each vessel were cutfrom each vessel, were mounted on end, and were examined byenvironmental scanning electron microscopy (ESEM). ESEM is performed onhydrated samples in relatively low vacuum. This permits thevisualization of the polymer film coating in the swollen and wet state.This is important to obtain measurements that may be readilyinterpreted, since the polymer film is approximately 95% water. A highdegree of thrombosis was readily observed in the control vessel. Thelumen of this vessel was narrowed to less than one-third its diameterpre-perfusion by the accumulation of thrombus, By contrast, no thrombuscould be observed in the lumen of the treated vessel, A highermagnification of the vessel wall demonstrated no adherent thrombun. Astill higher magnification shows a white structure which is the polymerfilm, which is different in contrast from the tissue due to differentialcharging under the electron beam of the ESEM. The film may be seen to beprecisely conformed to the shape of the vessel and be approximately 5-8μm thick.

The region of polymerization was restricted to the neighborhood of theblood vessel wall surface. The photosensitive dye was adsorbed to thevessel wall. Unbound dye was rinsed away. The entire lumen was filledwith prepolymer, but upon illumination the gel formation was restrictedto the vessel wall where the dye and the prepolymer meet. Thisinterfacial polymerization process can be conducted to produce surfaceadherent layers that vary in thickness from less than 7 μm to more than500 μm.

The above procedure was performed in 8 control rat arteries, and 8treated arteries, with equivalent light microscopic histological resultsas described above. As demonstrated by this study, PEG prepolymers canbe polymerized upon the lumenal surface of blood vessels. The immediateeffect of this modification is to reduce the thrombogenicity of aninjured blood vessel surface This has clear utility in improving theoutcome of balloon angioplasty by reducing the thrombogenicity of thevessel and lesion injured by balloon dilation. Another effect of thismodification is to be reduce smooth muscle cell hyperplasia. This may beexpected for two reasons. First, platelets contain a potent growthfactor, platelet-derived growth factor (PDGF), thought to be involved inpost-angioplasty hyperplasia. The interruption of the delivery of PDGFitself poses a pharmacological intervention, in that a “drug” that wouldhave been delivered by the platelets would be prevented from beingdelivered. Thrombosis results in the generation of thrombin, which is aknown smooth muscle cell mitogen. The interruption of thrombingeneration and delivery to the vessel wall also poses a pharmacologicalintervention. There are other growth factors soluble in plasma which areknown to be smooth muscle cell mitogens. The interruption of thrombingeneration and delivery to the vessel wall also poses a pharmacologicalintervention. Moreover, there are other growth factors soluble in plasmawhich are known to be smooth muscle cell mitogens. The gel layer isknown to present a permselective barrier on the surface of the tissue,and thus the gel layer may reasonably be expected to reduce hyperplasiaafter angioplasty. The inhibition of thrombosis upon the vessel wall mayalso reduce the incidence of abrupt reclosure and vasospasm, both ofwhich occur sometimes following vascular intervention.

EXAMPLE 21 Interfacial Polymerization of Macromers Inside Blood Vesselsto Prevent Thrombosis

Macromer solutions were polymerized interfacially within previouslyinjured blood vessels in vivo to prevent thrombosis. The carotid arterywas exposed, and a polyethylene tube (PE-10) was used to cannulate theexterior carotid artery. The artery was clamped with fine arterialclamps proximal to the interior/exterior carotid artery bifurcation andapproximately 2 cm distal to the bifurcation. A 1 ml tuberculin syringewas used to rinse the blood from the lumen of the isolated zone byfilling and emptying the vessel zone. The vessel was injured by crushingusing a hemostat. The isolated zone was filled with a 10 mM solution ofeosin Y for 2 minutes, after which it was rinsed and filled with a 20%solution of a macromer in saline with 0.1 mM triethanolamine and 0.15%N-vinyl pyrrolidinone. The macromer consisted of a PEG chain of MW 8,000daltons, extended on both sides with a lactic acid oligomer of anaverage degree of polymerization of 5 lactidyl groups, and furtheracrylated nominally at both ends by reaction with acryloyl chloride. Thevessel was illuminated transmurally using an argon ion laser (514 nm) atan intensity of approximately 1 mW/cm² for 5 seconds. Following this,the cannula was removed from the exterior carotid artery and the arterywas ligated at the bifurcation. The-arterial clamps were removed topermit the resumption of blood flow. Perfusion was allowed for 20minutes, following which the vessel were again isolated, removed fromthe body, gently rinsed, fixed, and prepared for light microscopichistological analysis. Using the naked eye, the crushed segments incontrol animals, which lacked illumination, were red, indicatinginternal thrombus with entrapped red blood cells. By contrast, noredness was observed at the site of the crush injury in the treatedvessels. Histology showed extensive thrombus, fibrin, and entrapped redblood cells in the non-treated vessels. By contrast, no thrombus orfibrin or entrapped red blood cells were observed in the treatedvessels. The procedure was conducted in four control animals and threetreated animals.

This example demonstrates that the polymerization can be carried out insitu in the living animal, that the polymer coating remains adherent tothe vessel wall during arterial blood flow, and that the polymer coatingcan prevent thrombosis in vivo in non-anticoagulated animals. Thisapproach to treatment has clear benefits in preventing abrupt reclosure,vasospasm, and restenosis after intravascular interventional procedures.Moreover, it is more generally applicable to other intraluminal andopen-surface organs to be treated.

Modifications and variations of the present invention, the macromer andpolymeric compositions and methods of use thereof, will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended

We claim:
 1. A biodegradable polymer comprising one or more macromerscomprising at least one water soluble region, at least one degradableregion, and at least two free radical polymerizable regions, wherein thepolymerizable regions are separated from each other by at least onedegradable region.
 2. The polymer of claim 1, wherein the water solubleregion is selected from the group consisting of poly(ethylene glycol),poly(ethylene oxide), poly(vinyl alcohol), poly(vinyl pyrrolidone),poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propyleneoxide) blockcopolymers, polysaccharides, carbohydrates, proteins, and combinationsthereof.
 3. The polymer of claim 1, wherein the degradable region isselected from the group consisting of poly(hydroxyl acids),poly(lactones), poly (amino acids), poly(anhydrides), poly(orthoesters), poly (phosphazines), and poly(phospboesters).
 4. Thepolymer of claim 1, wherein the polymerizable regions arephotopolymerizable regions.
 5. The polymer of claim 1, wherein the watersoluble region is attached to a degradable region.
 6. The polymer ofclaim 1, wherein the polymer is a hydrogel.
 7. The polymer of claim 1,wherein the polymer is a tissue support or sealant.
 8. The polymer ofclaim 7, wherein the tissue support or sealant is a shaped article. 9.The polymer of claim 8, wherein the shaped article is in a form selectedfrom the group consisting of microspheres, sheets, rods, and particles.10. The polymer of claim 1, further comprising a biologically activeagent.
 11. The polymer of claim 10, wherein the biologically activeagent is selected from the group consisting of proteins, carbohydrates,nucleic acids, organic molecules, inorganic biologically activemolecules, cells, tissue, and tissue aggregates.
 12. The polymer ofclaim 1, wherein the polymer has a total water content ranging fromabout 82% (w/w) to 94.5% (w/w).